拟轮枝镰孢丙氨酸转氨酶FvALT的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0432

摘要/Abstract

摘要：

【目的】拟轮枝镰孢是玉米穗腐病的主要病原菌，探究拟轮枝镰孢FvALT的生物学功能，为新型靶向杀菌剂开发提供理论依据。【方法】利用ProtParam、ProtScale、WoLF PSORT和Clustal X等在线工具对FvALT的生理生化特征及同源性进行分析；采用SOMPA和SWISS-MODEL获得其蛋白结构，并利用AutoDock分析FvALT与底物的结合能力；进而通过原核表达和亲和镍柱层析方法获得目的蛋白，通过荧光滴定技术验证FvALT与底物的结合能力。【结果】FvALT为亲水性蛋白并定位于胞浆中，其蛋白二级结构以α-螺旋和无规则卷曲为主，并在镰孢菌中高度保守，具有典型的PTZ00377 superfamily结构域；FvALT的最佳诱导条件为0.4 mol/L IPTG，16℃诱导16 h；FvALT与α-酮戊二酸具有较强的结合作用，结合位点为ARG³¹⁰、SER¹⁴⁹、SER²⁹⁸、SER³⁰⁰和ASP²⁵⁸。【结论】拟轮枝镰孢FvALT具有典型的丙氨酸转氨酶特征，与底物α-酮戊二酸特异结合。

关键词: 拟轮枝镰孢, 丙氨酸转氨酶, 生物信息学分析, 原核表达, 分子对接

Abstract:

【Objective】 Fusarium verticillioides is the main pathogen of maize tassel rot, thus exploring the biological function of FvALT will provide a theoretical basis for the development of novel targeted fungicides. 【Method】Physiological and biochemical characteristics and homology of FvALT were analyzed using online tools such as ProtParam, ProtScale, WoLF PSORT and Clustal X. The protein structure was obtained using SOMPA and SWISS-MODEL, and the binding ability of FvALT to substrates was analyzed using AutoDock. The target proteins were then obtained by prokaryotic expression and affinity nickel column chromatography, and the binding ability of FvALT to the substrate was verified by fluorescence titration technique.【Result】FvALT is a hydrophilic protein and localized in the cytoplasm, its protein secondary structure is dominated by α-helix and irregular coiling, and is highly conserved in Fusarium, with a typical PTZ00377 superfamily structural domain. The optimal conditions for the induction of FvALT are 0.4 mol/L IPTG, 16℃ for 16 h. FvALT has strong binding effects with α-ketoglutarate, and the binding sites are ARG³¹⁰, SER¹⁴⁹, SER²⁹⁸, SER³⁰⁰ and ASP²⁵⁸. FvALT has a strong binding effect with α-ketoglutarate, and the binding sites are ARG³¹⁰, SER¹⁴⁹, SER²⁹⁸, SER³⁰⁰, and ASP²⁵⁸.【Conclusion】FvALT has a typical alanine aminotransferase profile and binds specifically to the substrate alpha-ketoglutarate.

Key words: Fusarium verticillioide, FvALT, bioinformatics analysis, prokaryotic expression, molecular docking

郝楠, 耿珊, 赵雨薇, 侯智涵, 赵斌, 刘颖超. 拟轮枝镰孢丙氨酸转氨酶FvALT的克隆与表达分析[J]. 生物技术通报, 2024, 40(12): 256-263.

HAO Nan, GENG Shan, ZHAO Yu-wei, HOU Zhi-han, ZHAO Bin, LIU Ying-chao. Cloning and Expression Analysis of FvALT from Fusarium verticillioides[J]. Biotechnology Bulletin, 2024, 40(12): 256-263.

图/表 5

图1 拟轮枝镰孢FvALT蛋白高级结构 A：二级结构；B：三级结构；C：三级结构评估

Fig. 1 Advanced structure of FvALT protein of F. verticillioides A: Secondary structure. B: Tertiary structure. C: Evaluation of tertiary structure

图2 ALT蛋白序列比对及系统进化分析 A：真菌、植物和动物中的ALT蛋白的同源分析；B：真菌中ALT蛋白的氨基酸比对

Fig. 2 Protein sequence alignment and phylogenetic analysis of ALT A: Homologous analysis of ALT proteins in fungi, plants, and animals. B: Amino acid alignment of ALT proteins in fungi

图3 FvALT在大肠杆菌BL21（DE3）中的表达 M：蛋白质分子量标准；1：未经IPTG诱导菌株；2：经IPTG诱导裂解菌株沉淀；3：经IPTG诱导裂解菌株上清液； 4：流穿液；5：100 mmol/L咪唑洗脱；6：150 mmol/L咪唑洗脱

Fig. 3 Expression of FvALT in E. coli BL21（DE3） M: Standard of protein molecular weight. 1: Strains without IPTG induction. 2: Precipitation of lysed strain with IPTG induction. 3: Lyse the strain supernatant with IPTG induction. 4: Flow-through fluid. 5: 100 mmol/L imidazole elution. 6: 150 mmol/L imidazole elution

图4 拟轮枝镰孢FvALT与底物结合能力分析

Fig. 4 Analysis of the binding ability of FvALT to substrate in F. verticillioides

图5 拟轮枝镰孢FvALT与α-酮戊二酸的结合位点分析

Fig. 5 Analysis of the binding sites of FvALT in F. verticillioides and α-ketoglutarate

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